Elimination of methicillin-resistant Staphylococcus aureus biofilms on titanium implants via photothermally-triggered nitric oxide and immunotherapy for enhanced osseointegration.

BACKGROUND: Treatment of methicillin-resistant Staphylococcus aureus (MRSA) biofilm infections in implant placement surgery is limited by the lack of antimicrobial activity of titanium (Ti) implants. There is a need to explore more effective approaches for the treatment of MRSA biofilm infections. METHODS: Herein, an interfacial functionalization strategy is proposed by the integration of mesoporous polydopamine nanoparticles (PDA), nitric oxide (NO) release donor sodium nitroprusside (SNP) and osteogenic growth peptide (OGP) onto Ti implants, denoted as Ti-PDA@SNP-OGP. The physical and chemical properties of Ti-PDA@SNP-OGP were assessed by scanning electron microscopy, X-ray photoelectron spectroscope, water contact angle, photothermal property and NO release behavior. The synergistic antibacterial effect and elimination of the MRSA biofilms were evaluated by 2',7'-dichlorofluorescein diacetate probe, 1-N-phenylnaphthylamine assay, adenosine triphosphate intensity, o-nitrophenyl-ß-D-galactopyranoside hydrolysis activity, bicinchoninic acid leakage. Fluorescence staining, assays for alkaline phosphatase activity, collagen secretion and extracellular matrix mineralization, quantitative real­time reverse transcription­polymerase chain reaction, and enzyme-linked immunosorbent assay (ELISA) were used to evaluate the inflammatory response and osteogenic ability in bone marrow stromal cells (MSCs), RAW264.7 cells and their co-culture system. Giemsa staining, ELISA, micro-CT, hematoxylin and eosin, Masson's trichrome and immunohistochemistry staining were used to evaluate the eradication of MRSA biofilms, inhibition of inflammatory response, and promotion of osseointegration of Ti-PDA@SNP-OGP in vivo. RESULTS: Ti-PDA@SNP-OGP displayed a synergistic photothermal and NO-dependent antibacterial effect against MRSA following near-infrared light irradiation, and effectively eliminated the formed MRSA biofilms by inducing reactive oxygen species (ROS)-mediated oxidative stress, destroying bacterial membrane integrity and causing leakage of intracellular components (P < 0.01). In vitro experiments revealed that Ti-PDA@SNP-OGP not only facilitated osteogenic differentiation of MSCs, but also promoted the polarization of pro-inflammatory M1 macrophages to the anti-inflammatory M2-phenotype (P < 0.05 or P < 0.01). The favorable osteo-immune microenvironment further facilitated osteogenesis of MSCs and the anti-inflammation of RAW264.7 cells via multiple paracrine signaling pathways (P < 0.01). In vivo evaluation confirmed the aforementioned results and revealed that Ti-PDA@SNP-OGP induced ameliorative osseointegration in an MRSA-infected femoral defect implantation model (P < 0.01). CONCLUSIONS: These findings suggest that Ti-PDA@SNP-OGP is a promising multi-functional material for the high-efficient treatment of MRSA infections in implant replacement surgeries.

Situación nutricional en los primeros 1000 días del ciclo vital: una puesta al día de los datos disponibles en Argentina / Nutritional situation in the first 1000 days of life: an UpDate of the available data in Argentina

Introducción: Los primeros 1000 días de vida representan un período crítico del ciclo vital que impacta en el estado de salud de la mujer y de la progenie. Objetivo: Realizar una revisión sobre la información disponible con relación al estado nutricional materno-infantil, la lactancia materna y los hábitos alimentarios en esta etapa de la vida en el territorio de la Argentina. Métodos: Búsqueda de información de investigaciones realizadas en el país en niños y niñas de 0 a 2 años, embarazadas y mujeres en período de lactancia, publicadas en el período 2013-2020. Resultados: Los datos disponibles muestran que la alimentación tanto en las mujeres como en los niños y las niñas puede ser deficiente en nutrientes críticos, asociada a un perfil epidemiológico con prevalencia de sobrepeso. La baja talla, un indicador de malnutrición intergeneracional, se describe en una proporción de aproximadamente el 8% al 9% tanto en los menores de 2 años como en las gestantes, en tanto que la proporción de bajo peso se encuentra en el orden del 2 % al 10%, respectivamente. Los datos sobre lactancia materna revelan que aún no se alcanza a garantizar una cobertura satisfactoria; solo del 36,5% al 63,4% de los niños reciben lactancia materna exclusiva al sexto mes. La deficiencia de hierro y la anemia siguen siendo problemáticas frecuentes y en los pocos estudios regionales disponibles se encuentran resultados variables según la región y la población estudiada. Conclusión: Tanto los resultados de las encuestas nacionales como los procedentes de pequeños estudios poblacionales regionales muestran que las condiciones de vulnerabilidad social comprometen el estado nutricional durante estos primeros 1000 días de vida. Si bien se han implementado en el país en los últimos años una serie de programas e intervenciones de ayuda alimentaria, no existe información respecto del impacto que dichas políticas públicas han tenido en la población

Introduction: The first 1000 days of life represent a critical period of the life cycle that impacts the health status of women and their offspring. Objective: To carry out a review of the information available in relation to the maternal and infant nutritional status, breastfeeding and eating habits at this stage of life in the territory of Argentina. Methods: Search for information on research performed in Argentina on infants from 0 to 2 years of age, pregnant women and lactating women, published in the period 2013-2020. Results: The available data show that the diet of both women and children can be deficient in critical nutrients, associated with an epidemiological profile with prevalence of overweight. Short stature, an indicator of intergenerational malnutrition, is described in a proportion of approximately 8% to 9% in both those under 2 years of age and in pregnant women, while the proportion of underweight is in the order of 2% at 10% respectively. Data on breastfeeding reveal that satisfactory coverage has not yet been guaranteed; only 3,5% to 63,4% of children are exclusively breastfed at the sixth month. Iron deficiency and anemia continue to be frequent problems and the few available regional studies find variable results depending on the region and population studied. Conclusion: Both the results of national surveys and those from small regional population studies show that conditions of social vulnerability compromise nutritional status during these first 1,000 days of life. Although a series of food aid programs and interventions have been implemented in the country in recent years, there is no information regarding the impact that these public policies have had on the populatio

Genistein suppresses the proliferation of telomerase-negative cells.

In both tumor and yeast cells that lack telomerase, telomeres are maintained via an alternative recombination mechanism. In this study, we tested genistein, a potential TOP2 inhibitor required for telomere-telomere recombination, on the repression of telomere-telomere recombination. Genistein on the repression of type II recombination on a tlc1 yeast strain was examined by the telomeric DNA structures using Southern blot analysis. Telomere patterns of freshly dissected tlc1 spores containing an empty plasmid (pYES2) or a yeast TOP2 (yTOP2) plasmid were analyzed. The results indicated that the reintroduction of TOP2 recovered the type II pattern, implying genistein in the blockage of type II survivors in the tlc1 strain. The effects of genistein on both tlc1 and tlc1 rad 51 strains in liquid and solid mediums were also examined. Finally, treatment of 10 µmol/L of genistein showed inhibitory effect on the growth of telomerase-negative U2OS alternative lengthening of telomere (ALT) cells, but not in telomerase-positive HCT116 cells. These results provide evidences that the inhibitory effects of genistein on telomerase-negative cells depend on type II recombination pathway in yeast and the ALT pathway in human tumors.

Topoisomerase II inhibition suppresses the proliferation of telomerase-negative cancers.

Telomere maintenance is required for chromosome stability, and telomeres are typically elongated by telomerase following DNA replication. In both tumor and yeast cells that lack telomerase, telomeres are maintained via an alternative recombination mechanism. Previous studies have indicated that yeast Sgs1 and Top3 may work together to remove highly negative supercoils that are generated from recombination. However, the mechanism by which cells eradicate highly positive supercoils during recombination remains unclear. In the present study, we demonstrate that TOP2 is involved in telomere-telomere recombination. Disturbance of telomeric structure by RIF1 or RIF2 deletion alleviates the requirement for TOP2 in telomere-telomere recombination. In human telomerase-negative alternative lengthening of telomere (ALT) cells, TOP2α or TOP2ß knockdown decreases ALT-associated PML bodies, increases telomere dysfunction-induced foci and triggers telomere shortening. Similar results were observed when ALT cells were treated with ICRF-193, a TOP2 inhibitor. Importantly, ICRF-193 treatment blocks ALT-associated phenotypes in vitro, causes telomere shortening, and inhibits ALT cell proliferation in mice. Taken together, these findings imply that TOP2 is involved in the ALT pathway, perhaps by resolving the highly positive supercoil structure at the front of the helicase. Inhibition of topoisomerase II may be a promising therapeutic approach that can be used to prevent cell proliferation in ALT-type cancer cells.

Functional analysis of unfermented and fermented citrus peels and physical properties of citrus peel-added doughs for bread making.

Several studies have indicated citrus peels (CP) contain specific methoxy flavones, e.g. nobiletin and tangeretin, which have been shown to prevent numerous diseases. However, research reports regarding their application as food additive in healthy baked products is scarce. In our study, both unfermented (UF) and fermented (F) citrus peels were processed under different dry hot-air temperatures to make four citrus peel powders , UF-100 °C,UF-150 °C, F-100 °C, F-150 °C, respectively. The analysis of the basic components and nutraceuticals as well as antioxidant activity were conducted. Various percentages of CP were added to dough and toast bread for physical property and sensory evaluations. The results indicated the contents of crude proteins (3.3-4.3 mg/g) and fibers (10.9-14.9 %) among the four samples were similar. The UF extracts showed better antioxidant activities than F extracts. HPLC analysis indicated the contents of hesperidine, nobiletin and tangeretin in CP extracts were UF-150 °C > UF-100 °C. Farinograph analysis indicated a linear relation between CP powder content and the parameters of the physical properties of dough. A high percentage of fibrous CP powder in dough increases the water adsorption capacity of the dough, resulting in a decrease in its stability The sensory evaluation results indicated a greater acceptability of UF-added toast bread relative to the F-added one. Among these, according to the statistical anaylsis, the UF-150 °C 4 % and UF-100 °C 6 % groups were the best and F-150 °C 2 % group was the poorest in overall acceptability.

Preparation of curcumin microemulsions with food-grade soybean oil/lecithin and their cytotoxicity on the HepG2 cell line.

The choice of surfactants and cosurfactants for preparation of oral formulation in microemulsions is limited. In this report, a curcumin-encapsulated phospholipids-based microemulsion (ME) using food-grade ingredients soybean oil and soybean lecithin to replace ethyl oleate and purified lecithin from our previous study was established and compared. The results indicated soybean oil is superior to ethyl oleate as the oil phase in curcumin microemulsion, as proven by the broadened microemulsion region with increasing range of surfactant/soybean oil ratio (approx. 1:1-12:1). Further preparation of two formula with different particle sizes of formula A (30nm) and B (80nm) exhibited differential effects on the cytotoxicity of hepatocellular HepG2 cell lines. At 15µM of concentration, curcumin-ME in formula A with smaller particle size resulted in the lowest viability (approx. 5%), which might be explained by increasing intake of curcumin, as observed by fluorescence microscopy. In addition, the cytotoxic effect of curcumin-ME is exclusively prominent on HepG2, not on HEK293, which showed over 80% of viability at 15µM. The results from this study might provide an innovative applied technique in the area of nutraceuticals and functional foods.

Inhibitory effects of 1,3-bis-(2-substituted-phenyl)-propane-1,3-dione, ß-diketone structural analogues of curcumin, on chemical-induced tumor promotion and inflammation in mouse skin.

Dibenzoylmethane (DBM), a ß-diketone structural analogue of curcumin, has been reported to exhibit anti-tumorigenic and chemopreventive activities. Due to the structural resemblance of DBM to the anti-inflammatory curcumin and an aspirin-like skeleton of DBM derivatives, we tested the anti-inflammatory effects of DBM and its derivatives, 1,3-bis-(2-substituted-phenyl)-propane-1,3-dione, on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion as well as TPA- and arachidonic acid-induced mouse ear edema in skin of CD-1 mice. Topical application of 10 µmol DBM together with TPA on the back of mice previously treated with 7,12-dimethylbenz[α]anthracene (DMBA) inhibited TPA-induced skin tumor promotion significantly. In addition, 1,3-bis-(2-acetoxy phenyl)-propane-1,3-dione was a superior anti-inflammatory agent to aspirin (80% of inhibition), on TPA-induced mouse ear edema and reduced the production of prostaglandin E2 (PGE(2)), comparable to aspirin. Taken together, 1,3-bis-(2-acetoxyphenyl-propane-1,3-dione merits a valuable anti-inflammatory agent substituting aspirin in therapeutic treatment as well prevention of cancer.

Comparison of the antioxidant and transmembrane permeative activities of the different Polygonum cuspidatum extracts in phospholipid-based microemulsions.

The main purpose of this study was to investigate the transmembrane permeability of polyphenol-containing Polygonum cuspidatum extracts (PCE) encapsulated in phospholipid-based o/w microemulsion system. First, preparations of several PCEs using solid- or liquid-phase extraction or a combination of both, as well as evaluation of their antioxidant activities, were conducted and compared. In the antioxidant study, results indicated that PC-1 with the least extraction process exhibited the best antioxidant activity. By comparing the permeability coefficient (K(p)) among all tested PCEs in microemulsions (ME-PCs), ME-PC1 also possessed the largest permeability coefficients of both resveratrol and emodin. In addition, comparison of the transmembrane permeability of several polyphenol-encapsulated microemulsions showed that resveratrol had the most competitive advantage in the microemulsion formula for the control-release process. Taken together, it can be concluded that the matrix removed from the solid-phase extraction in PC-1 not only possesses antioxidant activity but also acts as an enhancer in transmembrane permeation. The structure specificity of the polyphenol plays important roles in the mechanism of the transmembrane permeation process. These findings might provide scientific evidence for the value of developing polyphenol-containing PCEs as nutraceuticals and cosmoceutical products.

Enhancement of the encapsulation and transmembrane permeation of isoflavone-containing red clover extracts in phospholipid-based microemulsions using different extraction processes.

Several isoflavone-enriched red clover extracts (RCE) prepared by using solid- or liquid-phase extraction or a combination of both were studied for their encapsulation into a phospholipid-based microemulsion system. The optimization process with regard to the bioactive ingredient-encapsulated amounts and transmembrane efficiency of various RCE micelles was demonstrated. The results indicated that the encapsulated amounts of isoflavones in RCE-encapsulated microemulsions (ME) of ME-RC5, ME-RC6, and ME-RC7 were increased by >10-fold when compared with that of the raw red clover extracts. Comparison of the permeability coefficient K(p) of the formononetin among the ME-RCs from the in vitro skin permeation study showed that ME-RC5 significantly exhibited the least permeability, whereas ME-RC6 exhibited enhanced permeability after two-stage solid-phase extraction, indicating the potential role of the matrix material as a barrier or enhancer in the transmembrane study.

Determination of the differential estrogenicity of isoflavonoids by E2-ER-ERE-dependent gene expression in recombinant yeast and MCF-7 human breast cancer cells.

The use of phytoestrogens-containing natural sources as alternative hormone replacement therapy (HRT) has been a subject of debate for decades. Development of assays to characterize these phytoestrogens is required. In this study, differential estrogenicities of five isoflavonoids found in red clover and soy, including biochanin A, daidzein, formononetin, genistein and glycitein were examined in a yeast-based screen system with a classical palindromic estrogen response element (ERE)-ADE2 reporter and in a MCF-7 cell culture system with mRNA levels of ER-dependent genes compared. In a yeast-based assay, five isoflavonoids showed various extents of estrogenic potencies. A collection of primary estrogen receptor (ER)-regulated genes by estradiol (E2), including hTERT, c-MYC, BCL2 and Ha-ras (oncogenic) and quinone reductase (QR), human complement 3 (C3) and COX7RP (non-oncogenic) were selected as marker genes for a MCF-7 cell-based endogenous gene expression assay. The results indicated that the mRNA levels of these E2-ER-ERE-dependent marker genes were regulated differentially by five isoflavonoids, leading to distinct expression patterns, which are also significantly different from that of E2. Moreover, the anti-estrogenic effects of biochanin A and formononetin on E2-induced transcriptions of marker genes in MCF-7 cells were also displayed. Taken together, these results are significant for these naturally occurring isoflavonoids regarding the issues of safety and efficacy.

Telomere configuration influences the choice of telomere maintenance pathways.

Telomere maintenance is required for chromosome stability, and telomeres are typically replicated by the action of telomerase. In yeast cells that lack telomerase, telomeres are maintained by alternative type I and type II recombination mechanisms. Previous studies identified several proteins to control the choice between two types of recombinations. Here, we demonstrate that configuration of telomeres also plays a role to determine the fate of telomere replication in progeny. When diploid yeasts from mating equip with a specific type of telomeric structure in their genomes, they prefer to maintain this type of telomere replication in their descendants. While inherited telomere structure is easier to be utilized in progeny at the beginning stage, the telomeres in type I diploids can gradually switch to the type II cells in liquid culture. Importantly, the TLC1/tlc1 yeast cells develop type II survivors suggesting that haploid insufficiency of telomerase RNA component, which is similar to a type of dyskeratosis congenital in human. Altogether, our results suggest that both protein factors and substrate availability contribute to the choice among telomere replication pathways in yeast.

Inhibition of estradiol-induced mammary proliferation by dibenzoylmethane through the E2-ER-ERE-dependent pathway.

The phytochemical dibenzoylmethane (DBM) has been shown to inhibit 7,12-dimethylbenz[a]anthracene induced mammary tumorigenesis in Sencar mice. However, the molecular basis of this activity is still elusive. In the present study, we demonstrated that DBM inhibits estradiol (E2)-induced incorporation of bromodeoxyuridine into mammary DNA in immature female Sencar mice by 52%, when 10 micromol of DBM was intraperitoneally injected into mice prior to the injection of E2. Examination of the influence of DBM on the expressions of E2-ERE-dependent oncogenes in MCF-7 cells indicated that DBM inhibits the E2-induced cell growth as well as the expressions of four oncogenes, telomerase, c-myc, Ha-ras and bcl-2. Further mechanistic study using chromatin immunoprecipitation assay demonstrated that DBM acts as a pure antagonist by attenuating the binding of estrogen receptor to the estrogen response elements in the regulatory regions of c-myc, hTERT and bcl-2 genes in vivo. Taken together, our results strongly suggest that DBM plays an inhibitory role in E2-induced proliferations, which establishes DBM as a model molecule for studying the antiestrogenic drugs.

Extrachromosomal telomeric circles contribute to Rad52-, Rad50-, and polymerase delta-mediated telomere-telomere recombination in Saccharomyces cerevisiae.

Telomere maintenance is required for chromosome stability, and telomeres are typically replicated by the telomerase reverse transcriptase. In both tumor and yeast cells that lack telomerase, telomeres are maintained by an alternative recombination mechanism. By using an in vivo inducible Cre-loxP system to generate and trace the fate of marked telomeric DNA-containing rings, the efficiency of telomere-telomere recombination can be determined quantitatively. We show that the telomeric loci are the primary sites at which a marked telomeric ring-containing DNA is observed among wild-type and surviving cells lacking telomerase. Marked telomeric DNAs can be transferred to telomeres and form tandem arrays through Rad52-, Rad50-, and polymerase delta-mediated recombination. Moreover, increases of extrachromosomal telomeric and Y' rings were observed in telomerase-deficient cells. These results imply that telomeres can use looped-out telomeric rings to promote telomere-telomere recombination in telomerase-deficient Saccharomyces cerevisiae.

Involvement of replicative polymerases, Tel1p, Mec1p, Cdc13p, and the Ku complex in telomere-telomere recombination.

Telomere maintenance is required for chromosome stability, and telomeres are typically replicated by the action of the reverse transcriptase telomerase. In both tumor and yeast cells that lack telomerase, telomeres are maintained by an alternative recombination mechanism. Genetic studies have led to the identification of DNA polymerases, cell cycle checkpoint proteins, and telomere binding proteins involved in the telomerase pathway. However, how these proteins affect telomere-telomere recombination has not been identified to date. Using an assay to trace the in vivo recombinational products throughout the course of survivor development, we show here that three major replicative polymerases, alpha, delta, and epsilon, play roles in telomere-telomere recombination and that each causes different effects and phenotypes when they as well as the telomerase are defective. Polymerase delta appears to be the main activity for telomere extension, since neither type I nor type II survivors arising via telomere-telomere recombination were seen in its absence. The frequency of type I versus type II is altered in the polymerase alpha and epsilon mutants relative to the wild type. Each prefers to develop a particular type of survivor. Moreover, type II recombination is mediated by the cell cycle checkpoint proteins Tel1 and Mec1, and telomere-telomere recombination is regulated by telomere binding protein Cdc13 and the Ku complex. Together, our results suggest that coordination between DNA replication machinery, DNA damage signaling, DNA recombination machinery, and the telomere protein-DNA complex allows telomere recombination to repair telomeric ends in the absence of telomerase.